Despite evidence that the tendency to develop obesity in humans is inherited and like to be polygenic in nature, the individual genes contributing to this neuroendocrine predisposition are largely unknown. Several mutations in exhibiting obesity phenotypes represent targets for the so-called "reverse genetic" approach which requires no knowledge or assumptions concerning the biological nature of the defect and seeks ultimately to identify genes based solely on their chromosomal location. The specific aims of this project are(1) to isolate additional anonymous DNA markers closely linked to the db locus, (2) provide more detailed resolution to the genetic linkage map of the db gene locus on mouse chromosome 4, (3) to derive the physical map of the db locus, (4) to examine the db locus and two other autosomal mutant gene loci that lead to obesity in the mouse for evidence of structural genomic DNA abnormalities, and (5) to begin studies ultimately to isolate these obesity genes. The explicit approaches will include the isolation of anonymous DNA markers from a phage lambda genomic library created from a hybrid cell line containing mouse chromosome 4 as its only mouse-derived genetic material, the deletion and mapping analysis of a panel of irradiation-reduced hybrid cell lines, RFLP analysis of interspecific and recombinant inbred mouse crosses, and separating and analyzing very large DNA molecules by pulsed field gel electrophoresis (PFGE) and blotting. Genomic DNA preparation (including samples in agarose plugs suitable for PFGE analysis) from mouse tissues and cell lines will be isolated. Coventional DNA Southern blots and genetic linkage experiments will be performed to provide more probes and mapping resources intended to narrow the focus on mouse chromosome 4 to identify the db gene. Blot transfers of PGFE experiments will be hybridized with various mouse and human DNA probes which are known to map closely to the db mutation and the other obesity mutations in mice. The examination of these large scale Southern blots will focus on (1) providing a physical genetic map for a complex of mutations near the db locus and (2) identifying potential structural abnormalities in DNA from db/db mice, mutants mapping near the db locus on mouse chromosome 4, and other single gene obesity mutants mapping to different but accessible mouse chromosomal loci. The long term objective are ultimately to identify the genes responsible for these genetic autosomal obesity syndromes. This grant proposal is of medical significance because any insights gained into these mouse mutations may help identify genes in mice and potential homologues in man contributing to the inherited tendency to develop obesity.